Part:BBa_K1935019
DNMT3a/DNMT3l
DNA (cytosine-5)-methyltransferase 3A: Required for genome-wide de novo methylation and is essential for the establishment of DNA methylation patterns during development. DNA methylation is coordinated with methylation of histones. It modifies DNA in a non-processive manner and also methylates non-CpG sites. May preferentially methylate DNA linker between 2 nucleosomal cores and is inhibited by histone H1. Plays a role in paternal and maternal imprinting. Required for methylation of most imprinted loci in germ cells. Acts as a transcriptional corepressor for ZBTB18. Recruited to trimethylated 'Lys-36' of histone H3 (H3K36me3) sites. Can actively repress transcription through the recruitment of HDAC activity.
DNA (cytosine-5)-methyltransferase 3-like: Catalytically inactive regulatory factor of DNA methyltransferases. It is essential for the function of DNMT3A and DNMT3B. Activates DNMT3A and DNMT3B by binding to their catalytic domain. Accelerates the binding of DNA and AdoMet to the methyltransferases and dissociates from the complex after DNA binding to the methyltransferases. Recognizes unmethylated histone H3 lysine 4 (H3K4) and induces de novo DNA methylation by recruitment or activation of DNMT3.
In vertebrates, Transcription Start Sites (TSS) are divided into two categories:
On the one hand, there are specific consensus sequences, such as the TATA box for example, that are recognized by transcription factors that recruit in turn RNA polymerases.
On the other hand, there are about 1 kb large DNA sequences with a high content of cytosine and guanine dinucleotides.
This last group is named “CpG promoters” or “CGIs” (CpG islands). It is the one which is targeted by DNA methyltransferases. Indeed, these regions are typically non-methylated, compared to the rest of the genome.
In addition to their CG abundance, CGIs show a particular histone modification signature: they are enriched in H3K4me3.This characteristic has consequences: it maintains DNMTs inactive. Indeed, DNA methyltransferases get activated only when bound to H3K27me3 via their ADD domain. That may explain why CGI are most of the time unmethylated.
Repression of transcription starts with recognition of deoxycytosines by the CXXC domain of histone modification-enzymes that trimethylate lysine 27 of histone H3. Thereafter, DNMTs are activated by the interaction of H3K27me3 with this ADD domain, as previously mentioned. We say DNA methylation acts like a lock because it acts at a higher level than histone-modification enzymes.
Fusing a DNAse-dead Cas9 (dCas9) to the DNMT enzymatic core won’t need H3K24me3 to be recruited because it is deleted for the ADD domain. In fact, it will be recruited in a sequence-specific way via sequence homology of a single guide (sg) RNA with sites chosen by the experimentator.
Before us, Albert Jeltsch’s team engineered a single chain (sc) fusion protein of catalytic core of murine Dnmt3a and activator domain of Dnmt3l which has a 7-fold increased activity in vitro. With this fusion protein fused to a TALEN, another team managed to methylate about 20% of the targeted CpG promoter and to reduce by one third the targeted gene transcription level.
The simplest idea is to directly fuse the sc DNMT3a::DNMT3l from Jeltsch to an inactive Cas9.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 1464
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